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. 2005 Jan;79(2):764-70.
doi: 10.1128/JVI.79.2.764-770.2005.

Mutations conferring resistance to human immunodeficiency virus type 1 fusion inhibitors are restricted by gp41 and Rev-responsive element functions

Daisuke Nameki  1 , Eiichi Kodama, Mieko Ikeuchi, Naoto Mabuchi, Akira Otaka, Hirokazu Tamamura, Mutsuhito Ohno, Nobutaka Fujii, Masao Matsuoka
Affiliations

Mutations conferring resistance to human immunodeficiency virus type 1 fusion inhibitors are restricted by gp41 and Rev-responsive element functions

Daisuke Nameki et al. J Virol. 2005 Jan.

Abstract

One of the human immunodeficiency virus (HIV) envelope proteins, gp41, plays a key role in HIV fusion. A gp41-derived peptide, T-20, efficiently inhibits HIV fusion and is currently approved for treatment of HIV-infected individuals. Although resistant variants have been reported, the mechanism of the resistance remains to be defined. To elucidate the mechanism in detail, we generated variants resistant to C34, a peptide derived from the gp41 carboxyl terminus heptad repeat (C-HR) in vitro. The resistant variants had a 5-amino-acid deletion in gp120 and a total of seven amino acid substitutions in gp41. Binding assays revealed that an I37K substitution in the N-terminal heptad repeat (N-HR) impaired the binding of C34, whereas an N126K substitution in the C-HR enhanced the binding to mutated N-HR, indicating that both mutations were directly involved in resistance. On the other hand, substitutions for A30 and D36 seemed to be secondary mutations, located complementary to each other in the Rev-responsive element (RRE), and were mutated simultaneously to maintain the secondary structure of the RRE that was impaired by the mutations at I37. Thus, HIV acquired resistance to C34 by mutations in N-HR, which directly interacted with C34. However, since this region also encoded the RRE, additional mutations were required to maintain viral replication. These results suggest that HIV fusion is one of the attractive targets for HIV chemotherapy.

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Figures

FIG. 1.
FIG. 1.
Schematic view of HIV-1 gp41 (A) and induction of C34-resistant HIV-1 (B). The locations of the fusion peptide (FP), N-terminal heptad repeat region (N-HR), C-terminal heptad repeat region (C-HR), transmembrane domain (TM), various gp41-derived peptides, and the Rev-responsive element (RRE) coding region are shown (A). The residue numbers of each peptide correspond to their positions in gp41. The bold underlined letters in the boxes indicate the novel mutations that have been reported in T-20-resistant HIV-1 variants (T-20r) in vitro (9, 26) and that have been observed in C34-resistant HIV-1 variants (C34r). (B) HIV-1NL4-3 was passaged in the presence of increasing concentrations of C34 in MT-2 cells. The dose-escalating selection was carried out for a total of 93 passages, with compound concentrations ranging from 0.0001 to 20 μΜ. At the indicated passages, proviral DNAs from the lysates of infected cells were sequenced, and the EC50s of the HIV-1 variants were determined with the MAGI assay.
FIG. 2.
FIG. 2.
Replication kinetics of the resistant variants. The replication kinetics determined by p24 antigen production and the CHRA are summarized. The data are depicted as the resistance (x axis) and replication (y axis) compared with those of HIV-1WT. Variants observed (continuous arrows) and not observed (dashed arrows) in the selection are shown in the order of their emergence.
FIG. 3.
FIG. 3.
Putative secondary structure of the RRE and locations of the nucleotides corresponding to the substitutions. The IIA, IIB, and IIC stems and the effects of the nucleotide substitutions are shown (A). A30V (GCC to GTC) to D36 (GAT) and A30 (GCC) to D36G (GAT to GGT) are located complementary to each other in the stem IIB (underlined). The effects of the nucleotide substitutions on the Rev-RRE interaction were examined by gel shift assays (B). The RRE of HIV-1I37T and HIV-1I37K displayed two signals. The amounts of Rev used were 0, 20, 40, 80, 160, 240, and 320 nM (left to right).
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